Evaluation dopamine receptors in Parkinson's

描述(申请人提供)：帕金森病(PD)是黑质纹状体多巴胺能神经元变性的结果。这个过程最终导致多巴胺介导的纹状体信号的进行性减少，表现为以运动迟缓、僵硬、震颤和步态异常为特征的临床综合征。帕金森病的传统治疗方法是通过注射多巴胺前体L-多巴来恢复纹状体中的多巴胺水平。对于晚期疾病，L-多巴会导致运动障碍和运动活动的显著波动(开关效应)。减轻这些副作用一直是一个重大挑战，并促使人们寻找替代策略，以提供更稳定的多巴胺能信号水平。为了恢复纹状体的多巴胺能活动，同时潜在地避免长期服用L-多巴的后果，一个以前未被探索的选择是通过引入结构性活性的多巴胺受体。PI的实验室在通过引入激活点突变来产生具有配体独立(或构成)活性的受体方面拥有丰富的经验。即使在没有多巴胺和/或多巴胺能激动剂药物的情况下，这些受体也有可能维持多巴胺能信号。这一应用的前提是，可以通过体外实验鉴定具有结构性活性的多巴胺受体，并在大鼠纹状体表达，以在更长的时间间隔内增强多巴胺能信号。特异性目标1的目的是在体外产生一系列具有结构性活性的多巴胺1和多巴胺2受体，并对其进行药理鉴定。利用重组腺相关病毒，将在大鼠身上探索纹状体过度表达固有活性的多巴胺受体的功能后果(特定目标2)。单侧病毒注射后的打圈行为将被用作构建活性的指标。所使用的方法将包括分子(成分活性突变受体的产生，重组蛋白的表达)，药理学(放射性配基结合，第二信使信号分析)和行为方法(循环行为的评估)。这些实验将进一步深入了解纹状体中多巴胺能受体的作用，并有可能为开发帕金森氏病的新治疗方案迈出第一步。

DESCRIPTION (provided by applicant): Parkinson's disease (PD) results from the degeneration of nigrostriatal dopaminergic neurons. This process ultimately leads to a progressive decrease in dopamine mediated striatal signaling which manifests as a clinical syndrome characterized by bradykinesia, rigidity, tremor, and gait abnormalities. Traditional therapy for PD has aimed at restoring dopamine levels in the striatum through administration of the dopamine precursor, L-dopa. With advanced disease, L-dopa leads to dyskinesias and periods of marked fluctuation in motor activity ('on-off effect'). Alleviation of these side effects has been a major challenge and has prompted a search for alternative strategies which can provide a more stable level of dopaminergic signaling. A previously unexplored option to restore striatal dopaminergic activity and at the same time to potentially avoid the consequences of long term L-dopa administration, is through the introduction of constitutively active dopamine receptors. The laboratory of the PI has extensive experience in generating receptors with ligand independent (or constitutive) activity through the introduction of activating point mutations. These receptors have the potential to maintain dopaminergic signaling even in the absence of dopamine and/or dopaminergic agonist drugs. The premise of this application is that constitutively active dopamine receptors can be identified using in vitro assays and expressed in the striatum of rats to enhance dopaminergic signaling over an extended time interval. The objective of Specific Aim 1 is to generate and pharmacologically characterize in vitro a series of constitutively active dopamine 1 and dopamine 2 receptors. Using recombinant adeno-associated virus, the functional consequences of striatal overexpression of constitutively active dopamine receptors will be explored in rats (Specific Aim 2). Circling behavior after unilateral viral administration will be used as an index of construct activity. The methodologies utilized will include molecular (generation of constitutively active mutant receptors, expression of recombinant proteins), pharmacologic (radioligand binding, second messenger signaling assays), and behavioral approaches (assessment of circling behavior). These experiments will provide additional insight into the role of dopaminergic receptors in the striatum as well as potentially take the first steps toward the development of a new therapeutic option for Parkinson's disease.